Currently, the existing technology commonly used in the industry is as follows: volumetric imaging can achieve quantitative and global measurement of complex systems, and is crucial in the research of cell metabolism, brain function and developmental biology. The simplest way to achieve volumetric imaging is to collect two-dimensional images with laser scanning in two-dimensional plane by means of optical sectioning scanning, so that the laser focus moves along the axial direction to obtain three-dimensional image information. In this method, high ability of the laser beam focus axial sectioning is required. Both confocal fluorescence microscopy and coherent Raman scattering microscopy have this ability. However, these sectioning methods have very strict requirements for sample size and are time-consuming for samples with thickness of several hundred microns. Light sheet microscopy overcomes the problem of time-consumption by broadening the laser beam into a thin plane, then scanning the sample by the thin plane and collecting images in a direction which is at 90 degrees to the direction of the laser beam to realize three-dimensional imaging of the entire volume of the sample. Light sheet fluorescence microscopy has achieved high resolution and high-speed volumetric imaging of biological samples from single cells to whole embryos. Although this method has been widely used, the fluorescent labels used in the imaging process may cause some serious problems, such as strong disturbance of biological system function, nonspecific targeting, cytotoxicity, etc. Raman light sheet microscopy based on spontaneous Raman effect can perform label-free volumetric imaging and avoid the problems caused by fluorescent labeling. However, in light sheet microscopy technology, the image quality usually degenerates with the distance from the sample surface to the objective lens. Another method of volumetric imaging is tomography, which collects transmission projection images of samples at different angles and reconstructs three-dimensional volume information by using angle-related images. Optical Projection Tomography (OPT) can produce isotropic high-resolution images of three-dimensional samples by light transmission or fluorescence emission. However, there is no contrast of chemical compositions in OPT of light transmission, and OPT for fluorescence emission is also limited to the problem of fluorescent labeling. Spontaneous Raman tomography was proposed by fusion of spontaneous Raman imaging with diffuse optical tomography, which can perform three-dimensional imaging of chemical compositions of samples, but its spatial resolution is low and the imaging speed is relative slow. In order to solve this problem, based on Bessel beam, stimulated Raman projection tomography is proposed by combining stimulated Raman scattering microscopy and projection tomography technology, which can achieve label-free volumetric imaging with micron-scale resolution and better speed. However, this method is unable to achieve a better three-dimensional imaging effect for large scale samples and provide the structural image information of samples. Although there are many methods available to achieve volumetric imaging of samples, these methods are either limited by the problem of fluorescent labeling or imaging performance, and these methods can only provide single information of the structure or chemical compositions, and cannot obtain multi-mode image information at the same time.
To sum up, the existing technology has the following problems: the existing technology for achieving volumetric imaging of samples is limited by fluorescent labeling and imaging performance, which can only provide single information of the structure or chemical compositions, and cannot obtain multi-mode image information at the same time. 1. The influence of fluorescent labels. The fluorescent labels used in the current imaging process may cause some serious problems, such as strong disturbance of biological system function, nonspecific targeting, cytotoxicity, etc.; 2. the imaging scale is small, and currently the way capable of label-free three-dimensional imaging is generally only a few hundred microns; 3. the imaging process only obtains single information. Either the structural images can be obtained and the functional changes of molecules cannot be seen, or the functional images without labels can be obtained, but the structure and location information cannot be determined.
Difficulty and significance of solving the above technical problems are as follows:
Difficulty: how to obtain the fusion image of the structure and chemical compositions of large scale samples at the same time with a label-free manner;
Significance: impacts of fluorescent labels on biological systems can be avoided by label-free way; large scale imaging method can be used to image micron-scale samples to obtain images with micron resolution; at the same time, the structure and chemical compositions imaging ensure that the high fit fusion of the two kinds of information, so as to obtain more comprehensive information of the sample.